Project Summary Hsp70 is a universally conserved molecular chaperone that performs a variety of functions in the cell including protein folding of both newly synthesized and denatured proteins, protein transport across membranes and disaggregation of oligomerized proteins. Research has primarily focused on how Hsp70 function specificity arises through regulation of a) expression of Hsp70, b) isoform differences in the Hsp70 protein family and c) the variety of co-chaperone proteins that bind to the Hsp70 molecule. Despite the identification of several phosphorylation sites on both yeast and mammalian Hsp70 through global proteomic screens, the biological function of these remains unclear. All organisms require correct coordination of the cell cycle to grow and proliferate. Misregulation of cell cycle progression can result in either cell inviability or cancer. Progression of the cell from G1 to G2 requires the activity of cyclin-dependent kinase (CDK) CDK4. The critical activity of CDK4 is regulated by binding to cyclin proteins such as Cyclin D1, which rises and falls in abundance periodically through the cell cycle. Because of the important role of Cyclin D1 in cell cycle control, misregulation of Cyclin D1 activity (through increased transcription or stability) is often observed in cancer cells. Any strategy that lowers Cyclin D1 activity in cells may form the basis of novel anticancer therapies. Our recent studies have determined that phosphorylation of a single site on Hsp70 regulates chaperone function by altering both co-chaperone and client protein interactions. In particular, we have shown that increased Hsp70 phosphorylation promotes Cyclin D1 destruction. In this proposal, we expect to gain further mechanistic insight into how Hsp70 phosphorylation can alter cell cycle progression by downregulating the levels of Cyclin D1 and other important cell cycle proteins. We propose to use both molecular biology and state-of-the-art mass spectrometric techniques in breast cancer cells to achieve the aims of the objectives in our proposal. Understanding the regulation of Hsp70 phosphorylation in mammalian cells will provide us with a completely novel way to target chaperone activity. Hsp70 activity may be suppressed using specific phosphatase/kinase inhibitors. It may be possible to target specific `client' proteins though alteration of Hsp70 phosphorylation status and specific Hsp70 phospho-species may have a higher susceptibility to inhibitors. The scope of this work has broad implications for a variety of diseases associated with both the cell cycle and molecular chaperone function, including many types of cancer and neurodegenerative illnesses caused by protein aggregation (Huntington's disease, Alzheimer's disease and Creutzfeld-Jakob disease).